Laboratory tests for the diagnosis of liver disease

David A. Williams, Jan Rothuizen

1.4.3.1 Introduction

Definitive diagnosis of liver disease is often problematic. Many diseases lead to secondary hepatic changes (Table 1.9) and routine biochemical tests may reveal evidence of liver disease in animals that appear clinically normal.

Once primary hepatic disease is suspected, it is extremely im­portant that clinical suspicion is directed by the overall clinical picture and all available data, not just the results of selected laboratory tests in isolation. It is often informative to monitor changes in observed abnormalities at 2 to 4 week intervals, particularly when test results are equivocal. Over such a time interval nonspecific changes may abate, secondary changes of­ten remain fairly consistent depending on the primary disease process, and abnormalities associated with primary liver di­sease will often become more apparent. If the test results are initially equivocal and the clinical signs are vague, sequential evaluation may be necessary to allow time for the disease to be fully expressed.

Definitive diagnosis may ultimately require hepatic biopsy, but even histopathology may not provide a clear diagnosis since histological abnormalities may be patchy, diagnostic criteria are not well standardized between pathologists, and the size and quality of biopsy samples is often sub-optimal. It should be noted that a manual on the standardization of hepatic di­seases in small animals has recently become available.⁴ This manual also describes standards for histopathological evalua­tion of hepatic biopsies and is the result of the efforts of the WSAVA standardization group for hepatic diseases.

Table 1.9: Diseases associated with secondary hepatic abnormalities

■ Hyperadrenocorticism (dogs)

■ Adrenal overproduction of sex hormones (dogs)

■ Drugs

-phenobarbital (dogs)

-corticosteroids (dogs)

■ Hyperthyroidism (cats)

■ Hypoxia

-autoimmune hemolytic anemia

-shock

■ Chronic small intestinal disease

■ Acute pancreatitis

■ Diabetes mellitus

■ Periodontal disease

■ Sepsis

1.4.3.2 Routine hematological testing, urinalysis, and fecal examination

There are few alterations in blood cells that suggest hepatobil­iary disease. Most are changes in erythrocytes associated with fragmentation or changes in cell size or membrane composi­tion. Microcytosis with normochromia or slight hypochromia is a rather common finding in dogs with congenital portosys­temic shunts (≥60%); it is less common in cats with conge­nital portosystemic shunts (≥30%). Most of these animals are not anemic. The cause of microcytosis is poorly understood. Regardless of the mechanism, delay in attaining the full com­plement of hemoglobin causes erythrocytes to undergo an extra cell division, resulting in smaller than normal mature cells. The erythrocyte indices normalize after successful sur­gery to correct the portosystemic shunt. If there is also non- regenerative anemia, microcytosis must be distinguished from anemia of chronic disease (which may also include liver di­sease) causing microcytosis and relative iron deficiency, or from iron deficiency caused by chronic gastrointestinal blood loss.

Strongly regenerative anemia, with low hematocrit (gravity ≤1.025), presence of bilirubin in the urine of cats, and ammonium biurate crystalluria. In dogs, ex­cessive bilirubinuria may precede the onset of hyperbilirubi­nemia and jaundice.

Measurement of urinary urobilinogen has traditionally been used to assess the presence of extrahepatic bile duct obstruc­tion. However, there are so many confounding factors (e.g., influences by the intestinal flora, renal function, urine pH and specific gravity, and the exposure of the urine sample to light) that the test is now considered without diagnostic value and obsolete.

Acholic feces characterized by absence of stercobilins (fecal pigments) and steatorrhea are very rarely seen in patients with severe, usually extrahepatic, cholestasis. Severe hemolysis caus­ing increased bilirubin production and excretion may cause orange-colored feces.

1.4.3.3 Analysis of ascites fluid

If abdominal fluid is detected, a sample should be collected for laboratory analysis. In dogs with chronic liver disease causing intrahepatic portal hypertension, the ascitic fluid is a clear and colorless pure transudate. It contains very few cells (correlate well with the degree or severity of hepatic damage.

Both the chronicity of increased activities and the degree of elevation, as well as the overall clinical picture, should be taken into consideration when evaluating the significance of abnor­mal serum ALT activities. It is important to note that severe hepatic disease can be present in patients with a normal or minimally increased serum enzyme activity. Therefore, finding such values should not preclude further investigation, espe­cially if there are clinical signs or other laboratory evidence that suggest hepatobiliary disease. Especially with chronic liver diseases, when there is less hepatocellular damage per time unit, serum ALT activities may not be severely increased, yet the disease ultimately causes severe loss of hepatic functional capacity. On the other hand, acute diseases, during which

many cells are affected and release enzymes in a short time period, usually are associated with very high elevations of liver enzyme activities in serum. Owing to the large functional re­serve of the liver, hepatic functional capacity is usually not severely affected in such cases. The above considerations ex­plain why it is important to assess both liver enzyme activities and liver function tests for screening purposes. A good combi­nation to confirm or exclude liver diseases is serum ALT activ­ity with SBA concentrations (see below). If both are within the reference range, the chance that there is clinically signi­ficant liver disease is very low (it is concentrated tenfold. Cholecys­tokinin, released from the small intestine after a meal, is the main trigger for gall bladder contraction, which is a slow and gradual process. Conjugated bile acids in the small intestine facilitate fat absorption by emulsifying the fat.

Plasma ammonia concentration is a very sensitive and specific indicator of ammonia detoxification by the liver. As for many other hepatic functions, the liver has a huge reserve capacity for this function, so that increased ammonia or decreased BUN concentrations are rarely due to parenchymal liver dys­function, but more commonly caused by portosystemic shun­ting. Virtually all ammonia is formed in the intestinal tract and reaches the liver through the portal blood supply. If portal blood bypasses the liver (i.e., due to congenital or acquired portosystemic shunting), it reaches the systemic circulation and becomes increased. The effect of shunting on BUN is much smaller, as the decreased portal blood supply of the liver induces an increased hepatic arterial blood flow. Much of the systemic ammonia-rich blood reaches the liver via the arterial route and is converted into urea. Blood ammonia concentra­tion is, therefore, preferentially increased in patients with con­genital and acquired portosystemic shunts, particularly after feeding, or after oral or rectal administration of ammonium sulfate (i.e., for the purpose of an ammonia tolerance test). Blood ammonia is also increased in patients with hepatic en­cephalopathy from other causes, and its assay can thus be use­ful to diagnose hepatic encephalopathy as a cause of neurolo­gical signs.

Plasma ammonia was formerly only available through specia­lized veterinary or human hospital laboratories.

If the sample is not analyzed immediately but is transported to a specialized off-site laboratory, the blood should be spun im­mediately in a refrigerated centrifuge, and the plasma should be put in a new pre-cooled tube and stored on ice. Cooled plasma can be stored for only 45 minutes prior to analysis, so that transport is only possible to a nearby facility. Hemolysis should be avoided because erythrocytes contain about three times the ammonia concentration of plasma. It is also impor­tant to avoid the possibility of contact of the blood sample with ammonia-containing contaminants such as cigarette smoke and body fluids such as sweat or saliva that may contain more ammonia than the sample. The use of vacuum tubes with a rubber stopper may help in preventing such contamination.

If ammonia is increased above 150 mg/dl (75 μmol∕L), this confirms that neurological signs are caused by hepatic ence­phalopathy, or that portosystemic shunting is present. How-

ever, in rare cases, and especially when there is only a low shunting fraction (e. g., due to mild congenital portal vein hy­poplasia), basal fasting ammonia may be within normal limits. In such cases, an ammonia tolerance test can be performed, and will confirm or exclude the presence of portosystemic shunting with certainty.

Oral administration of ammonium compounds can induce vomiting. A rectal test, in which 2 ml/kg of a 5% ammonium sulfate or chloride solution is given by enema and deposited as proximally into the colon as possible, is better tolerated. Blood ammonia is measured before and 30 min after adminis­tration of the ammonium salt. Plasma values after ammonium chloride administration in normal animals or animals with parenchymal or cholestatic liver diseases without portosys­temic shunting do not exceed a twofold increase over baseline concentrations. An exaggerated response indicates congenital or acquired portosystemic shunting. Possible rare exceptions include rare inborn errors of ammonia metabolism, cats with hepatic lipidosis, and animals with fulminant liver necrosis. Clinical experience has revealed little risk of aggravating he­patic encephalopathy during a rectal ammonia tolerance test.

Serum cholesterol concentration

Serum cholesterol concentration is often routinely included in serum biochemistry profiles by commercial laboratories but affords little useful information in the diagnosis of hepatobili­ary diseases. Increased cholesterol can be found in practically all cholestatic and parenchymal liver diseases, whereas low cholesterol may be present in dogs and cats with portosystem­ic vascular anomalies, which is, however, not diagnostic.

Serum glucose concentration

Hypoglycemia is an unusual finding in patients with liver di­sease and reflects very severe loss of hepatic function. It may be seen in patients with chronic liver disease with less than 20% functional hepatocytes left, or in acute fulminant hepati­tis, associated with severe necrosis of the liver. Dogs with con­genital portosystemic shunting may also show hypoglycemia, which is usually not very severe, but may become important when the patient is fasted before surgery. Also, some hepatic neoplasms may produce insulin-like proteins that can lead to hypoglycemia.

Serum electrolyte concentrations

Hypokalemia may be a risk factor for the development of hepatic encephalopathy and can be caused by renal and gastro­intestinal loss, reduced intake, and secondary hyperaldostero­nism. Hypokalemia may induce hypokalemic alkalosis, which is an important factor facilitating hepatic encephalopathy by promoting a shift of ammonia to non-ionized NH₃, which can readily diffuse membranes.

1.4.3.6 Abnormalities of coagulation parameters

The impaired synthesis of coagulation factors may lead to pro­longed clotting times or even bleeding episodes in rare in­stances. Coagulation abnormalities may occur in patients with liver disease because of either vitamin K malabsorption, re­duced hepatic synthesis of coagulation factors, or DIC as a consequence of the underlying primary disorder. It is perhaps most common to find a subtle prolongation ofaPTT (1.5 times normal), abnormal fibrin degradation products (10-40 μg∕ml or >40 μg∕ml), and variable fibrinogen concentrations (hepatic function.

Serum electrolyte abnormalities may occur in anorectic and hypoalbuminemic dogs and cats with congenital or acquired portosystemic shunting. Hypocalcemia is usually mild and an incidental finding associated with hypoalbuminemia.

1.4.3.8 Speciesdifferences

There are notable differences between cats and dogs regarding numerous aspects of hepatic structure and function, as well as the types and frequency of different diseases encountered in each species. Specific differences with regard to the evaluation of marker test results also exist. For example, in cats, serum ALP is not induced by adrenal steroids or other drugs, which is a common cause of increases in serum activities of this en­zyme in dogs. Also, both the hepatic concentration and half life of ALP in cats are low, such that increases should always be considered significant in this species. GGT is similar in its ori­gins to ALP and generally behaves similarly to ALP when there is hepatobiliary disease, although it may be less affected by cholestasis and drugs than ALP in dogs and more affected by cholestasis than ALP in cats. One notable observation is that cats with hepatic lipidosis, but not those with other hepatopa- thies, classically have very high serum ALP activities but nor­mal GGT activities. As mentioned above, bilirubinuria may be an indicator of hepatic disease. However, it is not uncommon for normal dogs to have mildly to moderately elevated urine bilirubin concentrations. In contrast, this is not true in cats, and therefore bilirubinuria in this species should always be consi­dered indicative of hepatic disease and investigated further.

??9 Key Facts

■ Serum alanine aminotransferase activity reflects hepatocellular damage but not hepatic function.

■ Increased serum alkaline phosphatase activities in cats nearly always indicate significant hepatic disease, but in dogs numerous extrahepatic diseases, such as hyperadrenocorticism or bone disease can cause abnormalities.

■ A pre- and postprandial serum bile acids concentration is the most clinically useful test to assess liver function in both dogs and cats.

■ Blood ammonia concentration can help identify patients with hepatic encephalopathy.

■ Results of hepatic laboratory testing must be evaluated in the light of the clinical picture and cannot be used in isolation to assess patients with suspected liver disease.

References

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